Stimulation of ornithine decarboxylase activity in rat tissues by growth hormone and by serum growth factor from rats infested with spargana of Spirometra mansonoides

Ornithine decarboxylase activity was studied in heart, kidney, liver, thymus, lung, spleen, skeletal muscle and fat of hypophysectomized rats after growth hormone treatment. A marked increase in enzyme activity was observed in kidney and liver, and a significant increase in heart and thymus at 4 h after injection of growth hormone. The kidney was the most responsive organ with an increase in activity of about 100 fold. The enzyme activity in kidney responded to a dose of 10 μg of growth hormone. Daily injection for 12 days raised activity only in the heart. Infestation for 6–13 days with spargana of Spirometra mansonoides, which also causes growth of hypophysectomized rats, increased enzyme activity in the heart and thymus. Intravenous injection of serum of hypophysectomized rats infested with spargana of Spirometra mansonoides caused a significant increase in the enzyme activity in liver and kidney after 4 h. Growth hormone and the serum growth factor of sparganosis seem to share the characteristic of causing an early increase in ornithine decarboxylase activity in rat tissues. The marked response in kidney and liver raises the possibility that these organs are the primary targets of both substances.     

Quantitative modulation of thyroid iodide peroxidase by thyroid stimulating hormone

The activity of rat thyroid iodide peroxidase fell to 8% of the normal value 48 hours after hypophysectomy. Rats given injections of thyroid stimulating hormone manifested an enzyme activity indistinguishable from that of the sham-operated animals. Cycloheximide prevented the thyroid stimulating hormone-induced restoration of the enzyme activity. The incorporation of ¹⁴C-leucine into the thyroid gland decreased gradually and reached two thirds of the sham-operated group by 48 hours after hypophysectomy. Thyroid stimulating hormone administration prevented this decrease, as observed for iodide peroxidase activity. Thyroidal RNA contents decreased also in hypophysectomized rats, thyroid stimulating hormone treatment prevented the reduction of RNA contents and no significant change was observed in thyroidal DNA contents. These data are consistent with the idea that protein biosynthesis is involved in thyroid stimulating hormone regulation of thyroidal iodide peroxidase and that the life span of the peroxidase is less than 48 hours.     

Lysosomal enzymes in the rat harderian gland are altered by either bromocriptine treatment or hypophysectomy and hormone replacement therapy

Four groups of adult male hypophysectomized rats were injected subcutaneously twice daily between 0800-0900 hr and 1600-1700 hr with either saline diluent, 150 micrograms sheep prolactin and/or growth hormone (GH); intact rats received either saline or 150 micrograms bromocriptine twice daily. After 4 days of treatment, lysosomal enzyme assays revealed significant elevations in both acid phosphatase and alpha-mannosidase enzyme activities in the Harderian glands of saline-injected hypophysectomized rats compared to those in intact controls. beta-Glucuronidase levels were depressed and hexosaminidase activity unaffected by hypophysectomy treatment alone compared to intact controls. Lysosomal enzyme activities in hypophysectomized animals treated with prolactin were not different from the hypophysectomized control animals. However, treatment with GH alone or in combination with prolactin had a significant inhibitory effect on beta-glucuronidase, hexosaminidase, and alpha-mannosidase enzyme activities in the Harderian gland of hypophysectomized animals. Bromocriptine treatment in intact rats only elevated acid phosphatase activity. In summary, the patterns of responses did not reveal a role for prolactin in the control of Harderian gland lysosomal enzyme activities by the pituitary. However, some of the influence on this target system may be exerted by growth hormone.     

Effects of corticotropin releasing factor on locomotor activity in hypophysectomized rats

Corticotropin releasing factor (CRF) injected intracerebroventricularly to hypophysectomized and sham hypophysectomized rats produced a dose dependent increase in locomotor activity, but in untreated hypophysectomized rats 10× more CRF was needed to produce a significant increase in activity. Concomitant daily supplements of rat growth hormone, thyroxine, and corticosterone to the hypophysectomized rats eliminated locomotor activity differences between the two groups. There was no statistically significant difference in locomotor response to either saline, 0.1 μg CRF, 1.0 μg CRF or 10.0 μg CRF in the group of animals receiving hormonal supplements. These results demonstrate that CRF can produce behavioral activation in rats independently of its effects on releasing hormones from the pituitary gland.     

Effect of diverse mammalian gonadotropins on estrogen and progesterone production by cultured rat granulosa cells

The ability of gonadotropins from six mammalian species to stimulate estrogen and progesterone production was investigated in granulosa cells of hypophysectomized estrogen-primed immature female rats. Granulosa cells were cultured for 2 days in the presence of delta 4-androstenedione (10(-7) M) with or without various gonadotropin preparations. Treatment with follitropin (follicle-stimulating hormone, FSH) from human, rat, ovine, porcine, equine, and bovine origins resulted in dose-dependent increases in steroidogenesis from negligible amounts to maximal levels of approximately 4-8 and 12-30 ng/10(5) cells for estrogen and progesterone, respectively. The ED50 values of the FSH preparations for stimulation of steroidogenesis were: human: 1-4 ng/ml; ovine: 2.5-30 ng/ml; rat: 1.6-4.0 ng/ml; porcine: 7.5-20 ng/ml; equine 2.5-6 ng/ml; and bovine greater than 100 ng/ml. Lutropin (luteinizing hormone, LH) from rat, ovine, bovine, and porcine origins, human chorionic gonadotropin (hCG), the alpha-subunit of human FSH and the beta-subunit of human LH were ineffective in stimulating steroidogenesis, indicating the specificity of the assay system for FSH. In a high concentration (600 ng/ml), the beta-subunit of human FSH-stimulated steroidogenesis to a small extent. Furthermore, pregnant mare serum gonadotropin and equine LH also caused a dose-dependent stimulation of estrogen and progesterone production, the half-maximal response values (ED50) being 1.8-4 and 7.5-10 ng/ml, respectively. This is consistent with previous in vivo and in vitro findings, showing the potent FSH activities of these hormones. Thus, the cultured rat granulosa cell system provides a sensitive assay for measuring FSH activities of gonadotropins from various mammalian species.     

Stimulation of fatty acid synthesis in vitro by gonadotrophin-induced testicular ribonucleic acid

RNA from testes of hypophysectomized rats treated with follicle-stimulating hormone and luteinizing hormone markedly stimulates in vitro the incorporation of acetate and malonate (as CoA derivatives) into polyunsaturated fatty acids. The system in vitro contains the components necessary for both protein and fatty acid synthesis. That the RNA is a hormone-induced messenger type that causes enzyme synthesis that then causes fatty acid synthesis is supported by the following observations: (1) the stimulation of RNA synthesis by follicle-stimulating hormone and luteinizing hormone is decreased by injection of the animals with actinomycin D; (2) puromycin in the system in vitro decreases the synthesis of polyunsaturated fatty acids; (3) the activity of the RNA preparation is destroyed by digestion with ribonuclease; in fact, the digest is inhibitory, which is a characteristic of messenger-RNA-mediated protein synthesis; (4) protein that might be denatured enzyme is virtually absent from the effective RNA preparations.     

Hormonal control of pulmonary surfactant synthesis

The specific activities of palmitoyl-CoA synthetase, phospholipase A2, and lysophosphatidylcholine acyltransferase enzymes were low in the lungs of diabetic and hypophysectomized rats as compared to those found in the normal controls. Administration of triiodothyronine (T3), to the diabetic and hypophysectomized rats restored the normal activities of these enzymes. Stimulation of the enzyme activities were also observed when normal rats were injected with the above hormone. The enhancement of the enzyme activities was also found to be dependent on the dose and duration of the hormonal treatment. Optimum levels were achieved at a dose of about 100 micrograms/100 g body weight of T3, 3-4 days after the administration of this hormone. Actinomycin D or cycloheximide abolished the hormone-mediated stimulation of these enzymes in diabetic and hypophysectomized rats. Reduced rate of in vivo palmitoyl-CoA synthetase synthesis was observed in the lungs of diabetic and hypophysectomized animals. Administration of T3 stimulated the rate of synthesis of this enzyme indicating increasing synthesis of this enzyme and not of activation of the pre-existing inactive species. Reduced phospholipid contents, specially decreased amount of lecithin and dipalmitoyl lecithin (DPL) were observed in the lungs of the diabetic and hypophysectomized animals as compared to those in the normal animals. T3 also increased the lecithin and DPL content of the normal rat lungs. These results provide evidence for the involvement of the thyroid hormones in the control of the pulmonary surfactant. The results further suggest that T3 was capable of inducing the enzymes of the "deacylation-reacylation" pathway involved in palmitate incorporation into phosphatidylcholine thereby contributing to the stimulation of dipalmitoyl phosphatidylcholine biosynthesis.     

Endocrine control of cytosolic factors stimulating adenylate cyclase in rat lung

Endocrine control of cytoplasmic factors modulating adenylate cyclase activity in rat lung membranes was investigated. Hypophysectomy, adrenalectomy and thyroidectomy showed an adverse effect on the body and organ weights. Lung protein, glycogen and DNA contents were decreased in the endocrine ablated animals which were restored to the normal values on hormone treatment. Phosphodiesterase and phosphorylase activities were increased and decreased in adrenalectomized and thyroidectomized animals, respectively. The activities of these enzymes were restored to normal values on hormone treatment. Adrenalectomy and thyroidectomy affected ATPases differently. Basal adenylate cyclase activity in rat lung membranes was not affected by adrenalectomy and hormone treatment. However, the total enzyme activity was increased by both dexamethasone (DEX) and thyroxine (T4) treatments. The activation of the particulate adenylate cyclase by the cytoplasmic factors was markedly decreased in the lung from hypophysectomized, adrenalectomized and thyroidectomized rats. This decrease in the cytoplasmic activation of adenylate cyclase was restored to or above the control values on hormone treatment. Alteration in the activation of enzyme by cytoplasmic factors did not appear to be due to the change in the responsiveness of the enzyme. Glucocorticoids appeared to have a specific effect on the cytoplasmic factors modulating the enzyme.     

Possible growth hormone regulation of rat liver glutamine synthetase activity.

Hypophysectomy results in a marked decrease in glutamine synthetase activity of rat liver homogenates. The enzyme is affected to a lesser extent in the kidneys and is not influenced in the brain. Bovine growth hormone treatment of hypophysectomized rats elevates the diminished glutamine synthetase activity in liver and kidneys but has no effect on the brain enzyme. Adrenalectomy also results in decreased liver glutamine synthetase activity although less than the decline seen with hypophysectomy. Cortisol treatment has no effect on glutamine synthetase activity in hypophysectomized animals. Our results suggest that growth hormone is involved in the regulation of liver glutamine synthetase activity. This regulation may be important in the utilization of α-amino nitrogen from glucogenic amino acids associated with growth hormone enhanced glucose production.     

Preparation and properties of biologically active radioiodinated parathyroid hormone

A constant-current microelectrolytic radioiodination method was used to label bovine parathyroid hormone (BPTH) with ¹²⁵I to an overall iodination ratio of 1:1 iodide atoms per PTH molecule. Such iodinated preparations were shown to be fully active in several bioassay systems: in vitro adenylate cyclase activation in rat renal and skeletal membranes, in vitro calcium release from rat calvaria, and the in vivo hypercalcemic response in chickens. Analysis by Sephadex G-15 chromatography after enzymatic digestion showed the radioiodine to be incorporated predominantly as monoiodotyrosine. Bioassay of iodinated preparations from which uniodinated hormone had been removed by isoelectric focusing showed essentially full hormonal activity. Such methods can be used to consistently produce radioiodinated biologically active preparations of BPTH 1–84 with high specific activity (2000 Ci/mmol).     

The effects of hypophysectomy and of growth hormone on the uptake of radioactive phosphorus by tissues

Normal and hypophysectomized animals, fed ad lib., both with and without growth hormone treatment, were injected with 4 microcuries of P³²/100 g. body weight, and then autopsied at varying time intervals following the injection.The plasma of hypophysectomized animals possesses a greater P³² activity than normal plasma at all intervals from 0.5 to 24 hr. after P³² injection. Maintenance of hypophysectomized animals with growth hormone results in a plasma P³²-activity level much smaller than that found in the plasma of hypophysectomized controls, yet greater than that found in the plasma of normal animals.The normal liver uptake of P³² shows a maximum between 0.5 and 1 hr. after intraperitoneal injection. Kidney shows a maximum in less than 0.5 hour, and thymus attains a maximum activity level in 2 hr. Hypophysectomy results in an increased liver, kidney, and thymus P³² uptake when compared to the normal. After hypophysectomy the time of maximal P³² activity in liver is shifted to 4 hr. after injection. Growth hormone therapy in the hypophysectomized animal lowers the P³²-uptake levels of kidney and liver toward normal, and brings down the P³² level in thymus to normal.Hypophysectomy with or without growth hormone therapy, results in a muscle P³² accumulation which is less than normal. Growth hormone administration to the normal animal causes an increased P³² accumulation by muscle.The P³² uptake by the tibia of the hypophysectomized rat is less than normal. Maintenance of the hypophysectomized rat with growth hormone prevents the decrease in bone P³² uptake due to hypophysectomy.     

Alteration of liver plasma membrane protein conformation by bovine growth hormone in vitro

The response of liver plasma membranes prepared from hypophysectomized, bovine growth hormone-treated hypophysectomized, and normal rats to addition of bovine growth hormone in vitro were studied by circular dichroism and spectrofluorometry. Membranes of hypophysectomized but not normal or treated rats showed increased negative ellipticity in the presence of bovine growth hormone (10^?9) without any change in trough position; at higher concentrations (10^?7?10^?6, m) there was less negative ellipticity. Membranes of hypophysectomized, normal, and growth hormone-treated rats all showed decreased emission of fluorescence and a small shift of the emission peak from 333 to 338 nm in the presence of bovine growth hormone (10^?17?10^?7, m). The excitation of fluorescence was quenched by bovine growth hormone. Polarization of excitation of fluorescence was unchanged.     

Renal phosphoenolpyruvate carboxykinase turnover in hypo- and hyperthyroid rat in vivo

The effect of hypo- and hyperthyroidism on activity, synthesis and degradation of renal cytosolic phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) was studied in the rat by radioimmunological techniques. In hypo- and euthyroid rats, starvation induced similar alterations in enzyme activities and relative rates of synthesis, whereas in hyperthyroid rats the increase in both was significantly reduced. Substitution of l-thyroxine in hypothyroid rats resulted in a decrease in activity and synthesis within 18 h as observed in hyperthyroid animals. The apparent half-life of the enzyme measured by double-pulse labeling experiments was approx. 13 h in euthyroid animals. The rate of degradation was unaffected by the different thyroid states.     

Lysyl oxidase: a pituitary hormone-dependent enzyme.

Aldehyde-deficient non-crosslinked collagen obtained from lathyritic rats and collagen from penicillamine-treated rats, which is not deficient in aldehydes but the crosslinking of which is also inhibited, were implanted into the peritoneal cavity of hypophysectomized rats using the diffusion chamber technique. The enzyme lysyl oxidase which catalyses the aldehyde formation in certain lysyl residues of collagen and elastin was extracted from the skin of hypophysectomized rats. The activity of the enzyme was determined following its incubation with an L-[4,5-3H] lysine-labeled elastin substrate prepared from aortas of 17-day-old chick embryos. The result showed that the aldehyde deficient collagen did not crosslink while in the hypophysectomized animal indicating the lack of active lysyl oxidase in the rats. The enzyme activity in the skin of hypophysectomized animals was markedly reduced as compared with the controls indicating directly the dependance of lysyl oxidase activity on pituitary gland hormones.     

Influence of thyroxine and luteinizing hormone on some enzymes concerned with lipogenesis in adipose tissue, testis and adrenal gland

The activities of hexokinase, citrate-cleavage enzyme, ;malic enzyme' and NADP-linked isocitrate dehydrogenase have been measured in the adipose tissue, testes and adrenals of normal rats, hypophysectomized rats and hypophysectomized rats treated with either thyroxine or thyroxine plus luteinizing hormone. Hypophysectomy reduced the activity of all four enzymes in all three tissues. Thyroxine alone restored the activity of all four enzymes in adipose tissue towards normal but failed to do so in either testes or adrenals. Thyroxine and luteinizing hormone restored the citrate-cleavage enzyme activity of testes and increased the activity of hexokinase from the low value after hypophysectomy. Neither ;malic enzyme' nor isocitrate dehydrogenase was increased by thyroxine or thyroxine and luteinizing hormone in testes. The differential stimulation of enzyme activity by thyroxine in the different tissues suggests thyroxine as having a special significance in adipose-tissue lipogenesis.     

Growth hormone and drug metabolism. Acute effects on microsomal mixed-function oxidase activities in rat liver.

Adult male rats were subjected either to sham operation or to hypophysectomy and adrenalectomy and maintained for a total of 10 days before treatment with growth hormone. Results of the early effects of growth hormone on the activities of the mixed-function oxidases in rat liver over a 96h period after growth-hormone treatment are presented. 2. Hypophysectomy and adrenalectomy result in decreased body and liver weight and decreased drug metabolism (mixed-function oxidases). Concentrations of electron-transport-system components are also decreased. 3. In the hypophysectomized/adrenalectomized rats, growth hormone decreases the activities of the liver mixed-function oxidases and the cytochrome P-450 and cytochrome c reductases, as well as decreasing the concentration of cytochrome P-450 compared with that of control rats. Similar but less dramatic results are obtained with sham-operated rats. 4. It is concluded that whereas growth hormone enhances liver growth, including induction of many enzyme activities, it results in a decrease in mixed-function oxidase activity. Apparently, mixed-function oxidase activity decreases in liver when growth (mitogenesis) increases.     

Increased activity of spermidine N1-acetyltransferase in the adrenal cortex of rats following administration of exogenous spermidine, carbamylcholine, 2-deoxyglucose and dopamine agonists

The activity of N1-acetyltransferase was increased in the dissected adrenal cortex of the rat following a single administration of spermidine. The activity was maximal 6-8 h after the onset of treatment. The increase in enzyme activity was abolished when the rats were given cycloheximide 2 h after spermidine; this suggests that increased activity results from an augmentation in the synthesis of the enzyme. Adrenocortical spermidine N1-acetyltransferase was also induced by carbamylcholine, 2-deoxyglucose, apomorphine and piribedil, drugs that are known to cause induction of ornithine decarboxylase in that organ. Hypophysectomized rats showed reduced activity of spermidine N1-acetyltransferase when compared to sham-operated controls, and carbamylcholine no longer elicited an increase in enzyme activity in such animals. Adrenocortical spermidine N1-acetyltransferase activity of hypophysectomized rats is induced by corticotropin (ACTH). These results suggest a hormonal control over the activity of the enzyme in the adrenal cortex with ACTH acting as a mediator.     

Growth hormone and drug metabolism. Acute effects on nuclear ribonucleic acid polymerase activity and chromatin.

Adult male rats, subjected either to sham operation or to hypophysectomy and adrenalectomy were maintained for 10 days before treatment with growth hormone. Results of the acute effects of growth hormone on the rat liver nuclear RNA polymerase I (nucleolar) and II (nucleoplasmic) activities as well as the chromatin template capacity were then studied and compared with the growth-hormone effects on the drug metabolism described in the preceding paper (Wilson & Spelsberg, 1976). 2. Conditions for isolation and storage of nuclei for maintenance of optimal polymerase activities are described. It is verified that the assays for polymerase activities require a DNA template, all four nucleoside triphosphates, and a bivalent cation, and that the acid-insoluble radioactive product represents RNA. Proof is presented that under high-salt conditions DNA-like RNA (polymerase II) is synthesized, and that under low-salt conditions in the presence of alpha-amanitin, rRNA (polymerase I) is synthesized. 3. In the livers of hypophysectomized/adrenalectomized rats, growth hormone increases the activity of both RNA polymerase enzymes and the chromatin template capacity within 1h after treatment. The effects last for 12h in the case of polymerase II but for only 6h in the case of polymerase I. Sham-operated rats respond to growth hormone in a manner somewhat similar to that shown by hypophysectomized/adrenalectomized rats. These results, which demonstrate an enhancement of RNA polymerase I activity in response to growth hormone, support those from other laboratories. 4. Growth-hormone enhancement of the chromatin template capacity in the liver of hypophysectomized/adrenalectomized rats contrasts with previous reports. The growth-hormone-induced de-repression of the chromatin DNA could represent the basis of the growth-hormone-induced enhancement of RNA polymerase II activity in the hypophysectomized/adrenalectomized rats, although some effect of growth-hormone on the polymerase enzymes is still suggested.